1. Field of Invention
The present invention relates to confocal microscopes and the use thereof, and more particularly to single-mode fiber-optic confocal microscopes.
2. Discussion of Related Art
High-resolution confocal laser microscopy is an emerging field in modern imaging and bioimaging technologies. This technique provides sharp, high-magnification, three-dimensional imaging with submicron resolution by non-invasive optical sectioning and rejection of out-of-focus information (see, T. Corle and G. Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems,” Academic Press, San Diego, 1996; P. Torok and M. Gu, “High-numerical aperture optical microscopy and modern applications: introduction to the feature issue”, Appl. Opt. 39, pp. 6277-6278, 2000; S. Kimura and T. Wilson, “Confocal scanning optical microscope using single-mode fiber for signal detection”, Appl. Opt. 30, pp. 2143-2150, 1991; P. Delaney, M. Harris and R. King, “Fiber-optic laser scanning confocal microscope suitable for fluorescence imaging”, Appl. Opt. 33, pp. 573-577, 1994). Depending on the fundamental principle of operation, the various confocal techniques are classified into three major groups: reflectance, fluorescence, and multi-photon confocal microscopes (see FIG. 1). The conventional confocal microscope has mainly been used in reflection mode, which is the operating principle as well of the ultrahigh-resolution confocal microscope approach of the present invention. While fluorescence and multi-photon confocal microscopy are based on a nonlinear conversion of the input laser wavelength, reflectance microscopy is linear, i.e., it is not related to any frequency conversion imaging technique, and thus it doesn't require special spectral filtering equipment. Three-dimensional reflectance imaging uses light reflected from a small, localized volume of an object, such as tissue, to form images. There are two main groups of reflectance confocal systems: pinhole-based and fiber-optic-based confocal microscopes (FIG. 1).
The conventional pinhole-based confocal microscope utilizes a bulk diffraction-limited optical design (see FIG. 2) in which both the light source (L) and the detector (D) usually take the form of micrometer-scale pinholes. The confocal principle involves the confocal microscope potential for both illumination of only one spot on the sample through the input pinhole (Ain) and imaging the radiation back reflected from the sample using a beam splitter (BS) to the output pinhole (Aout). In this way, the use of small pinholes reduces the amount of the scattered light and the signal-to-noise ratio increases by rejection of the out-of-focus signal. As a result, sharp high-magnification imaging of thick samples is obtained, which is one of the basic advantages of confocal microscopy over conventional optical microscopy. The optimum size and spacing of the pinhole depends on the focal length and numerical aperture of the confocal objectives (O). A larger pinhole transmits more light to the detector (D), generating a larger signal but less resolution. A smaller pinhole has theoretically better resolution, but transmits less light to the sample, so the signal-to-noise ratio decreases. In addition, the high-numerical-aperture objectives (NA>0.8) in confocal microscope arrangements provide high depth and spatial discriminations and thus, high axial and lateral resolutions are obtained, respectively (see, T. Corle and G. Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems,” Academic Press, San Diego, 1996). However, the bulk optical design with micrometer-sized pinholes that is usually used in conventional confocal systems has certain disadvantages related to unacceptable signal attenuation, diffraction and aberration effects, misalignment problems, inflexibility, and dust obstruction.
On the sub-wavelength nanometric scale, where there has been recently a great impetus to obtain quantitative chemical information at cellular and intracellular level, the optical imaging techniques have a major drawback related to their spatial resolution which results from the fundamental “Rayleigh diffraction resolution limit” that is theoretically one-half the wavelength of the operating radiation. In the case of confocal microscopy, the diffraction limit affects the out-of-focus light. A pinhole-based confocal microscope attempts to remove this light with a confocal pinhole aperture, but such an aperture would need to have a diameter close to zero to eliminate the out-of-focus contribution. An effective way to avoid the resolution limit and to work far beyond it is a fiber-optic-based confocal microscope approach as with the ultrahigh-resolution confocal microscope and methods of the current invention.
To improve the dynamic range of the resolving laser power and to achieve a high spatial resolution in the nanometric spatial range, recently the current inventors have suggested a novel design of a simple reflection-type confocal microscope with a single optical fiber output (see, Iev and R. Waynant, “A simple submicron confocal microscope with a fiber-optic output”, Rev. Sci. Instrum. 71, pp. 4161-4164, 2000; 1. Ilev and R. Waynant, “Submicron reflection confocal microscope with a multimode fiber output”, Conference on Lasers and Electro-Optics (CLEO-2000), Paper CThM77, San Francisco, 2000; 1. Ilev, R. Waynant, K. Byrnes and J. Anders, “Dual-confocal fiber-optic method for absolute measurement of refractive index and thickness of optically transparent media”, Opt. Lett. 27, pp. 1693-1695, 2002; the entire contents of which are hereby incorporated by reference). An experimental optical setup of this fiber-optic confocal design is shown in FIG. 3. It is an apertureless confocal arrangement having a pair of objective lenses (O1 and O2) and a beam splitter (BS), in which the micrometer-sized pinholes are replaced by a highly sensitive optical fiber. The laser emission is launched directly, without input pinhole, to the scanned object. In this way, all possible negative effects leading to signal attenuation such as reduction of the input laser power, need of additional collimation, diffraction and aberration effects are eliminated, and a regime of maximum input signal level is ensured. Moreover, the confocal design includes a graded-index 50.mu.m core diameter multimode fiber used for signal detection rather than a conventional pinhole. Because of much lower numerical aperture of the optical fiber than that of a conventional pinhole, the multimode fiber-optic output shows much higher sensitivity to spatial displacements of the focusing back reflected radiation than a pinhole.